Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis

Summary Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety Chinese Spring. From these and previous studies it is concluded that some , and gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE > 85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of Chinese Spring carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in Chinese Spring. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in Chinese Spring is shown to be a high molecular weight globulin.     

Genetics of gliadins coded by the group 1 chromosomes in the high-quality bread wheat cultivar Neepawa

Summary The inheritance and biochemical properties of gliadins controlled by the group 1 chromosomes of the high-quality bread wheat cultivar Neepawa were studied in the progeny of the cross Neepawa x Costantino by six different electrophoretic procedures. Chromosome 1B of Neepawa contains two gliadin loci, one (Gli-B1) coding for at least six - or -gliadins, the other (Gli-B3) controlling the synthesis of gliadin N6 only. The map distance between these loci was calculated as 22.1 cM. Amongst the chromosome 1A gliadins, three proteins are encoded at the Gli-A1 locus whereas polypeptides N14-N15-N16 are controlled by a remote locus which recombines with Gli-A1. Six other gliadins are controlled by a gene cluster at Gli-D1 on chromosome 1D. Canadian wheat cultivars sharing the Gli-B1 allele of Neepawa were found to differ in the presence or absence of gliadin N6. The electrophoretic mobilities of proteins N6 and N14-N15-N16 were unaffected by the addition of a reducing agent during two-dimensional sodium dodecyl sulphate polyacrylamid-gel electrophoresis, suggesting the absence of intra-chain disulphide bonds in their structure.Research supported by a grant from the Commission of the European Communities, ECLAIR programme, Contract AGRE 0052     

Production and genetic characterization of near-isogenic lines in the bread-wheat cultivar Alpe

Two biotypes of the bread-wheat cultivar Alpe were shown to possess contrasting alleles at each of the glutenin (Glu-B1, Glu-D1, Glu-B3 and Glu-D3) and gliadin (Gli-B1 and Gli-D1) loci on chromosomes 1B and 1D. Fourteen near-isogenic lines (NILs) were produced by crossing these biotypes and used to determine the genetic control of both low-molecular-weight (LMW) glutenin subunits and gliadins by means of one-dimensional or two-dimensional electrophoresis. Genes coding for the B, C and D groups of EMW subunits were found to be inherited in clusters tightly linked with those controlling gliadins. Southern-blot analysis of total genomic DNAs hybridized to a -gliadin-specific cDNA clone revealed that seven NILs lack both the Gli-D1 and Glu-D3 loci on chromosome 1D. Segregation data indicated that these null alleles are normally inherited. Comparison of the null NILs with those possessing allele b at the Glu-D3 locus showed one B subunit, seven C subunits and two D subunits, as fractionated by two-dimensional A-PAGExSDS-PAGE, to be encoded by this allele. Alleles b and k at Glu-B3 were found to code for two C subunits plus eight and six B subunits respectively, whereas alleles b and k at Gli-B1 each controlled the synthesis of two -gliadins, one and two -gliadins. The novel Gli-B5 locus coding for two -gliadins was shown to recombine with the Gli-B1 locus on chromosome 1B. The two-dimensional map of glutenin subunits showed -gliadins encoded at the Gli-A2 locus on chromosome 6A. The use of Alpe NILs in the study of the individual and combined effects of glutenin subunits on dough properties is discussed.Research supported by a grant from the Commission of the European Communities, ECLAIR programme, Contract AGRE 0052     

Dosage effects of chromosomes of homoeologous groups 1 and 6 upon bread-making quality in hexaploid wheat

Summary The endosperm storage proteins, glutenin and gliadin, are major determinants of bread-making quality in hexaploid wheat. Genes encoding them are located on chromosomes of homoeologous groups 1 and 6. Aneuploid lines of these groups in spring wheat cultivar Chinese Spring have been used to investigate the effect of varying the dosage of chromosomes and chromosome arms upon bread-making quality, where quality has been assessed using the SDS-sedimentation test. Differences between the group 1 chromosomes for quality were greater than those between the group 6 chromosomes. The chromosomes were ranked within homoeologous groups for their effect on quality as follows (>=better quality): 1D>1B>1A and 6A>6B=6D. The relationship of chromosome dosage with quality was principally linear for four of the chromosomes, but not for 6B and 6D. Increases in the dosage of 1B, 6A and, especially, 1D, were associated with significant improvements in quality, whereas increases in the dosage of 1A were associated with reductions in quality. The effects of 1A and 1D were such that the best genotype for quality was nullisomic 1A-tetrasomic 1D. For group 1, effects of the long arm appeared in general to be more important than effects of the short arm. For group 6, effects were found associated with the long arms as well as with the short arms, a surprising result in view of the absence of genes encoding storage proteins on the long arms. Significant interactions were found between chromosomes and genetic backgrounds, and between individual chromosomes. Analysis of trials grown over two years demonstrated that, although additive environmental differences over years and genotype x years interaction were present, they were relatively small in magnitude compared with purely genetic differences.     

RFLP patterns of gliadin alleles in Triticum aestivum L.: implications for analysis of the organization and evolution of complex loci

A correspondence between RFLP patterns and gliadin alleles at the Gli-1 and Gli-2 loci was established in a set of 70 common wheat (T.aestivum L.) cultivars using -gliadin (K32) and -gliadin (pTU1) specific probes. All Gli-B1 and Gli-D1 alleles which differed in encoded -gliadins showed definite RFLP patterns after hybridization with the K32 probe. Two groups of Gli-B1 alleles, Gli-B1b-like and Gli-B1e-like, were identified, and these could originate from distinct genotypes of the presumptive donor of the B-genome. Intralocus recombination and/or gene conversion as well as small deletions, gene silencing and gene amplification were assumed to be responsible for the origin of new gliadin alleles. Silent -gliadin sequences were shown to exist in all of the genotypes studied. K32 also differentiated Gli-A1a from all other Gli-A1 alleles as well as the Gli-B11 allele in cultivars carrying the 1B/1R (wheat/rye) translocation. PTU1 was shown to recognize several Gli-A2 alleles, but not the Gli-B2 or Gli-D2 alleles. Moreover, this probe hybridized to chromosome 1R sequences suggesting the existence of rye gene(s), probably silent, for -gliadin-like proteins on chromosome 1R.     

Distribution of 1AL.1RS and 1BL.1RS wheat-rye translocations in Triticum aestivum using specific PCR

Chromosome arm 1RS of rye (Secale cereale) is a valuable resource for wheat (Triticum aestivum) improvement. 1AL.1RS and 1BL.1RS translocations play an important role in wheat breeding, since wheat carrying these chromosomal translocations has higher tolerance to biotic and abiotic stress. In this study, the presence of 1RS and the distribution of 1AL.1RS and 1BL.1RS wheat-rye translocations were examined in 66 Iranian cultivars and 70 regional foreign accessions of bread wheat, using three rye-specific primers (“RYER3/F3”, “O-SEC5′-A/O-SEC3′-R”, “PAWS5/S6”). Based on “RyeR3/F3”, the presence of 1RS was verified in 15 (23%) Iranian cultivars and in two (3%) foreign accessions. Further, “O-SEC5′-A/O-SEC3′-R” and “PAWS5/S6” were used to distinguish 1AL.1RS and 1BL.1RS translocations. According to results from these primers, 1BL.1RS was identified in 14 (21%) Iranian cultivars and two (3%) foreign accessions. The results confirm that “Sholeh” is the only cultivar (1.5%), among all cultivars and accessions, that carries 1AL.1RS. This study provides a useful tool in marker-assisted selection of materials containing 1RS, and in the creation of new Iranian common wheat cultivars with a larger genetic diversity in wheat breeding programs.     

Electrophoretic analysis of the high-molecular-weight glutenin subunits of Triticum monococcum,T. urartu,and the A genome of bread wheat (T. aestivum)

Summary The high molecular weight (HMW) subunit composition of glutenin was analysed by sodium dodecyl sulphate, polyacrylamide gel electrophoresis (SDS-PAGE) in the A genome of 497 diploid wheats and in 851 landraces of bread wheat. The material comprised 209 accessions of wild Triticum monococcum ssp. boeoticum from Greece, Turkey, Lebanon, Armenia, Iraq, and Iran; 132 accessions of the primitive domesticate T. monococcum ssp. monococcum from many different germplasm collections; one accession of free-threshing T. monococcum ssp. sinskajae; 155 accessions of wild T. urartu from Lebanon, Turkey, Armenia, Iraq, and Iran; and landraces of T. aestivum, mainly from the Mediterranean area and countries bordering on the Himalayan Mountains. Four novel HMW glutenin sub-units were discovered in the landraces of bread wheat, and the alleles that control them were designated Glu-Ald through Glu-Alg, respectively. The HMW subunits of T. monococcum ssp. boeoticum have a major, x subunit of slow mobility and several, less prominent, y subunits of greater mobility, all of which fall within the mobility range of HMW subunits reported for bread wheat. In T. monococcum ssp. monococcum the range of the banding patterns for HMW subunits was similar to that of ssp. boeoticum. However, two accessions, while containing y subunits were null for x subunits. The single accession of Triticum monococcum ssp. sinskajae had a banding pattern similar to that of most ssp. boeoticum and ssp. monococcum accessions. The HMW subunit banding patterns of T. urartu accessions were distinct from those of T. monococcum. All of them contained one major x and most contained one major y subunit. In the other accessions a y subunit was not expressed. The active genes for y subunits, if transferred to bread wheat, may be useful in improving bread-making quality.     

Retrotransposon-based molecular markers for linkage and genetic diversity analysis in wheat

Retrotransposon-based molecular markers have been developed to study bread wheat ( Triticum aestivum) and its wild relatives. SSAP (Sequence-Specific Amplification Polymorphism) markers based on the BARE-1/ Wis-2-1A retrotransposons were assigned to T. aestivum chromosomes by scoring nullisomic-tetrasomic chromosome substitution lines. The markers are distributed among all wheat chromosomes, with the lowest proportion being assigned the D wheat genome. SSAP markers for BARE-1/ Wis-2-1A and three other wheat retrotransposons, Thv19 , Tagermina and Tar1, are broadly distributed on a wheat linkage map. Polymorphism levels associated with these four retrotransposons vary, with BARE-1/ Wis-2-1A and Thv19 both showing approximately 13% of bands polymorphic in a mapping population, Tagermina showing approximately 17% SSAP band polymorphism and Tar1 roughly 18%. This suggests that Tagermina and Tar1 have been more transpositionally active in the recent evolutionary past, and are potentially the more useful source of molecular markers in wheat. Lastly, BARE-1 / Wis-2-1A markers have also been used to characterise the genetic diversity among a set of 35 diploid and tetraploid wheat species including 26 Aegilops and 9 Triticum accessions. The SSAP-based diversity tree for Aegilops species agrees well with current classifications, though the Triticum tree shows several significant differences, which may be associated with polyploidy in this genus.Communicated by M.-A. Grandbastien     

Characterization of variability and relationships among components of partial resistance to leaf rust in CIMMYT bread wheats

Summary A study of spring bread wheat (Triticum aestivum) germ plasm developed at the International Maize and Wheat Improvement Center (CIMMYT) showed highly significant phenotypic variability for each component of partial resistance (namely, uredial appearance period, latency period, uredial number and uredial size) to Puccinia recondita f. sp. tritici. All of the wheat genotypes displayed longer uredial appearance and latency periods and decreased uredial number and uredial size when compared to the susceptible check cultivar Morocco. Positive correlations between uredial appearance period and latency period, and uredial number and uredial size, and negative correlations between uredial appearance and latency periods and uredial number and uredial size, inclusive, suggested that the components of partial resistance were either tightly linked or under pleiotropic genetic control. Compared to Morocco, all entries had slow disease progress in the field and variation occurred in the germ plasm for the area under the leaf rust progress curve. Disease progress was negatively correlated with uredial appearance and latency periods, whereas a positive correlation was observed with uredial number and uredial size. Certain genotypes displayed high levels of partial resistance resulting in low disease incidence in the field.     

Nitrate reductase activity (in vivo and in vitro) of ditelosomic stocks of wheat (Triticum aestivum L.)

The nitrate reductase activities (NRA) of 31 ditelosomic stocks were compared with that of the control plant [Chinese Spring (CS) euploid], using in vivo and in vitro assay procedures that had been optimized with respect to the euploid. Fourteen stocks exhibited significant differences in in vivo NRA from that of the euploid; the effect of removal of a chromosome arm was always to increase NRA. Eight of these stocks showed similar effects in vitro, although in three, a casein-sensitive factor had to be eliminated before the difference was expressed. Homoeologous group effects were evident among ditelosomics of groups 2, 4, and 7, while for three chromosomes (2D, 7A, and 7B), removal of either arm resulted in a similar increase in NRA in vivo and probably in vitro.P. W. Jones was supported by a Science Research Council C.A.S.E. award with the Plant Breeding Institute, Cambridge, U.K.     

Genetic linkage between endosperm storage protein genes on each of the short arms of chromosomes 1A and 1B in wheat

Summary The storage proteins of the endosperm of wheat grain which are known to be controlled by genes on the short arms of the homoeologous group 1 chromosomes are (1) the -gliadins, (2) most of the -gliadins, (3) a few -gliadins and (4) the major lowmolecular-weight subunits of glutenin. Several crosses were made between varieties or genetic lines which had contrasting allelic variants for some of these proteins and which were coded by genes on chromosomes 1A or 1B. The progeny were analysed by one or more of several electrophoretic procedures. The results of all the analyses are consistent with the hypothesis that chromosomes 1A and 1B each contain just one, complex locus, named Gli-A 1 and Gli-B 1 respectively, which contain the genes for the -, - and -gliadins and the low-molecular-weight subunits of glutenin.     

A storage-protein marker associated with the suppressor of Pm8 for powdery mildew resistance in wheat

A suppressor of resistance to powdery mildew conferred by Pm8 showed complete association with the presence of a storage-protein marker resolved by electrophoresis on SDS-PAGE gels. This marker was identified as the product of the gliadin allele Gli-A1a. The mildewresponse phenotypes of wheats possessing the 1BL.1RS translocation were completely predictable from electrophoretograms. The suppressor, designated SuPm8, was located on chromosome 1AS. It was specific in its suppression of Pm8, and did not affect the rye-derived resistance phenotypes of wheat lines with Pm17, also located in 1RS, or of lines with Pm7.     

Identification and molecular characterization of a large insertion within the repetitive domain of a high-molecular-weight glutenin subunit gene from hexaploid wheat

High-molecular-weight (HMW) glutenin subunits are a particular class of wheat endosperm proteins containing a large repetitive domain flanked by two short N- and C-terminal non-repetitive regions. Deletions and insertions within the central repetitive domain has been suggested to be mainly responsible for the length variations observed for this class of proteins. Nucleotide sequence comparison of a number of HMW glutenin genes allowed the identification of small insertions or deletions within the repetitive domain. However, only indirect evidence has been produced which suggests the occurrence of substantial insertions or deletions within this region when a large variation in molecular size is present between different HMW glutenin subunits. This paper represents the first report on the molecular characterization of an unusually large insertion within the repetitive domain of a functional HMW glutenin gene. This gene is located at the Glu-D1 locus of a hexaploid wheat genotype and contains an insertion of 561 base pairs that codes for 187 amino acids corresponding to the repetitive domain of a HMW glutenin subunit encoded at the same locus. The precise location of the insertion has been identified and the molecular processes underlying such mutational events are discussed.     

Production,morphology, and cytogenetic analysis of Elymus caninus (Agropyron caninum) x Triticum aestivum F1 hybrids and backcross-1 derivatives

Summary Intergeneric hybrids were produced between common wheat, Triticum aestivum (2n=6x=42, AABBDD) and wheatgrass, Etymus caninus (Agropyron caninum) (2n=4x=28, SSHH) — the first successful report of this cross. Reciprocal crosses and genotypes differed for percent seed set, seed development and F₁ hybrid plant production. With E. caninus as the pollen parent, there was no hybrid seed set. In the reciprocal cross, seed set was 23.1–25.4% depending upon wheat genotype used. Hybrid plants were produced only by rescuing embryos 12–13 days post pollination with cv Chinese Spring as the wheat parent. Kinetin in the medium facilitated embryo germination but inhibited root development and seedling growth. The hybrids were vigorous, self sterile, and intermediate between parents. These had expected chromosome number (2n=5x=35, ABDSH), very little chromosome pairing (0.51 II, 0.04 III) and some secondary associations. The hybrids were successfully backcrossed with wheat. Chromosome number in the BC₁ derivatives varied 54–58 with 56 as the modal class. The BC₁ derivatives showed unusually high number of rod bivalents or reduced pairing of wheat homologues. These were sterile and BC₂ seed was produced using wheat pollen.     

Genetic analysis of gliadin components in winter wheat using two-dimensional polyacrylamide gel electrophoresis

Summary Blocks of gliadin components found both in a number of varieties and in single F₂ grains of winter wheat intervarietal hybrids have been studied by two-dimensional electrophoresis combining electrophoresis in acidic aluminium-lactate buffer (pH3.1) and SDS-electrophoresis. Gliadin components (spots) have been shown to be inherited as linked groups (blocks), codominantly and in accordance with a gene dosage in triploid endosperm. Blocks include components differing in their electrophoretic mobility and molecular weight. Some allelic variants of blocks differ only in presence of few additional components or in the electrophoretic mobility of components with similar molecular weights; other variants may contain no similar components. Apparently, in the course of evolution, mutations in individual genes of gliadin-coding loci and processes changing the number of expressing genes and the sizes of their structural part occurred.     

Combining ability analysis over F1-F5 generations in diallel crosses of bread wheat

Summary Combining ability studies for grain yield and its primary component traits in diallel crosses involving seven diverse wheat cultivars of bread wheat (Triticum aestivum L.) over generations F₁-F₅ are reported. The general and specific combining ability variances were significant in all generations for all the traits except specific combining ability variance for number of spikes per plant in the F₅. The ratio of general to specific combining ability variances was significant for all the traits except grain yield in all the generations. This indicated an equal role of additive and non-additive gene effects in the inheritance of grain yield, and the predominance of the former for its component traits. The presence of significant specific combining ability variances in even the advanced generations may be the result of an additive x additive type of epistasis or evolutionary divergence among progenies in the same parental array. The relative breeding values of the parental varieties, as indicated by their general combining ability effects, did not vary much over the generations. The cheap and reliable procedure observed for making the choice of parents, selecting hybrids and predicting advanced generation (F₅) bulk hybrid performance was the determination of breeding values of the parents on the relative performance of their F₂ progeny bulks.     

Quantitative structure analysis of genetic diversity among spring bread wheats (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) from different geographical regions

Genetic diversity in spring bread wheat (T.␣aestivum L.) was studied in a total of 69 accessions. For this purpose, 52 microsatellite (SSR) markers were used and a total of 406 alleles were detected, of which 182 (44.8%) occurred at a frequency of <5% (rare alleles). The number of alleles per locus ranged from 2 to 14 with an average of 7.81. The largest number of alleles per locus occurred in the B genome (8.65) as␣compared to the A (8.43) and D (5.93) genomes, respectively. The polymorphism index content (PIC) value varied from 0.24 to 0.89 with an average of 0.68. The highest PIC for all accessions was found in the B␣genome (0.71) as compared to the A (0.68) and D␣genomes (0.63). Genetic distance-based method (standard UPGMA clustering) and a model-based method (structure analysis) were used for cluster analysis. The two methods led to analogical results. Analysis of molecular variance (AMOVA) showed that 80.6% of the total variation could be explained by the variance within the geographical groups. In comparison to the diversity detected for all accessions (H _e = 0.68), genetic diversity among European spring bread wheats was H _e = 0.65. A comparatively higher diversity was observed between wheat varieties from Southern European countries (Austria/Switzerland, Portugal/Spain) corresponding to those from other regions.     

Production and cytogenetic analysis of BC1, BC2, and BC3 progenies of an intergeneric hybrid between Triticum aestivum (L.) Thell. and tetraploid Agropyron cristatum (L.) Gaertn.

Summary Intergeneric hybrids between Triticum aestivum cv Chinese Spring and Agropyron cristatum 4x (2n= 5x=35, ABDPP genomes) with a high level of homoeologous meiotic pairing between the wheat chromosomes were backcrossed 3 times to wheat. Pollination of the F₁ hybrid with Chinese Spring resulted in 22 BC₁ seeds with an average seed set of 1.52%. Five BC₁ plants with 39–41 chromosomes were raised using embryo rescue techniques. Chromosome pairing in the BC₁ was characterized by a high frequency of multivalent associations, but in spite of this there was no evidence of homoeologous pairing between chromosomes of wheat and those of Agropyron. All of the plants were self sterile. The embryo rescue technique was again essential to produce 39 BC₂ plants with chromosome numbers ranging from 37 to 67. The phenomenon of meiotic non-reduction was also observed in the BC₃ progenies. In this generation male and female fertility greatly increased, and meiotic pairing was fairly regular. Some monosomic (2n=43) and double monosomic (2n=44) lines were produced. Analysis of these progenies should permit the extraction of the seven possible wheat-Agropyron disomic addition lines including those with the added chromosomes carrying the genes involved in meiotic non-reduction and in suppression of Ph activity.     

Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves

The present investigation was undertaken in order to select the surface-sterilization technique most efficient for eliminating epiphytes, to document the spectrum of endophytes of healthy leaves from three wheat cultivars in Buenos Aires Province (Argentina) and to determine their infection frequencies at three growth stages. Surface-sterilization with undiluted commercial solution of sodium hypochlorite was reaffirmed as adequate for removing epiphytes on wheat leaves. From the 450 wheat leaf segments incubated, three bacterial isolates and 130 fungal isolates were obtained. From all the isolates, 19 fungal species were identified. Bacterial isolates were characterized as Bacillus sp. There were significant differences between microorganisms, stages of growth, and stages × microorganisms interaction. Differences between cultivars, stages × cultivars, microorganisms × cultivars and for the triple interaction were not significant. Frequency of microorganisms isolated increased with crop age, but it was statistically similar for the three wheat cultivars tested (Klein Centauro, Klein Dragón and Buck Ombú). Rhodotorula rubra, Alternaria alternata, Cladosporium herbarum and Epicoccum nigrum were isolated in the highest frequency. The other microorganisms were present at intermediate or low values. The species isolated may be assigned to three groups: (a) well-known and economically important pathogens of wheat, (b) commonly abundant phylloplane fungi considered to be primary saprobic and minor pathogens and (c) species occasionally present in wheat.     

Detecting specific repeated sequences in large,complex genomes by using representative difference analysis and double-probe verification

We present a modification of the representative difference analysis (RDA) technique used to target AT-rich repeated sequences, such as transposable elements, with a double-probe verification system. RDA is a subtractive/amplification PCR-based technology used to identify specific sequences that are different between 2 related genomes.Vsp I restriction enzyme was used to target AT-rich sequences. RDA products were cloned with a high efficiency. Double-probe verification is based on reverse dot-blot of cloned RDA products and uses a positive and a negative probe. We tested thisVsp I-modified RDA on different combinations of bread wheat (Triticum aestivum) and relatives.Triticeae members have large, complex genomes with various ploidy levels. RDA experiments were performed with single or bulked DNA. Reverse dot-blot double-probe verification detected specific repeated sequences quickly and efficiently. Together, the 2 systems provide a powerful tool for obtaining specific transposable elements and repeated sequences that are different between related genomes, regardless of genome size and ploidy.